PROJECT SUMMARY The overall objective of this MIRA proposal is to understand the mechanisms through which eukaryotic stress response pathways are regulated by post-translational modification of proteins with the small ubiquitin-like modifier SUMO. Cells must respond and adapt to physical and/or chemical stresses that can irreversibly and lethally damage essential macromolecules. Stress-response pathways generally adjust transcription, translation, protein activity or interactions, and metabolism according to the particular stress and its severity. In eukaryotic cells, stress exposure leads to sumoylation of specific proteins; however, the role(s) of sumoylation during most stress responses still remain uncertain. This gap in our knowledge presents a key barrier to our understanding of dynamic cellular stress regulation and adaptation, and its functional importance in human health where stress signaling becomes strained or co-opted in various diseases (e.g. heart disease, cancer, neurodegeneration, type II diabetes). To investigate mechanisms by which protein sumoylation mitigates stress-related cellular damage, our research group has been using budding yeast as a model organism to identify specific roles for sumoylation during distinct stresses. Initially, we focused on hyperosmotic stress and discovered that the yeast prion protein Cyc8 and its binding partner Tup1 are rapidly and transiently sumoylated during hyperosmotic stress. In addition, we found that the Cyc8-Tup1 complex forms phase- separated nuclear foci during the initial stages of hyperosmotic stress, and Cyc8 sumoylation is important for the timely resolution of these foci during cellular adaptation to the stress. We hypothesize that the Cyc8-Tup1 complex's transient coalescence into liquid-liquid phase separations (LLPS) during hyperosmotic stress alters the complex's interactions with chromatin, allowing for the optimal expression of hyperosmotic stress-response genes. In parallel to the studies on hyperosmotic stress, we have continued exploring the role of sumoylation in other stresses. Thus, the MIRA mechanism is ideal for our continuing studies due to its flexibility to pursue timely and salient avenues of inquiry at the key intersection of two rapidly-evolving fields: stress-dependent sumoylation and the kinetics of LLPS formation. Here, we propose to use a combination of genetics, cell biology, and in vitro biochemistry to uncover both overarching principles for the functions of sumoylation across multiple stresses and specific roles for sumoylation during distinct stresses. Our goals over the next five years are to explore the following questions: 1) What are the functional purposes for multivalent sumoylation within a complex during specific stress responses? 2) Are there general principles for the function(s) of sumoylation across divergent stresses? 3) Through what mechanism(s) does sumoylation modulate stress-induced LLPS dynamics?